Anti-infective compound

ABSTRACT

The invention relates to novel infective agents, the use thereof for the production of a pharmaceutical composition for the treatment and prophylaxes of a disease, preferably an infectious disease, a pharmaceutical composition comprising said compound, and to methods of producing said compounds. The invention further relates to a new probiotic configured for preventing or reducing the colonization by a pathogenic microorganism of an organ of a living being.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation in-part of international patent applicationPCT/EP2016/056358 filed on 23 Mar. 2016 and designating the U.S., whichhas been published in English, and which claims priority from Europeanpatent application EP 15 160 285.1 filed on 23 Mar. 2015. The entirecontents of these prior applications are incorporated herein byreference.

REFERENCE TO A SEQUENCE LISTING

This application contains reference to amino acid sequences and/ornucleic acid sequences which have been submitted concurrently herewithas the sequence listing text file “5402P541USCONWO.txt”, file size 40KiloBytes (KB), created on 15 Dec. 2017. The afore-mentioned sequencelisting is hereby incorporated by reference in its entirety pursuant to37 C.F.R. § 1.52(e)(5).

FIELD OF THE INVENTION

The invention relates to a novel anti-infective compound, the usethereof for the production of a pharmaceutical composition for thetreatment and/or prophylaxis of a disease, preferably an infectiousdisease, a pharmaceutical composition comprising said compound, and tomethods of producing said compounds.

BACKGROUND OF THE INVENTION

The control and treatment of infectious diseases is one of the biggestchallenges of the modern society. Nearly 40,000 men, women and childrenare dying every day from infectious diseases worldwide.

RELATED PRIOR ART

Among the infectious agents pathogenic bacteria are more than ever ofhigh relevance. Bacterial infections may be treated with antibiotics,which are intended to kill the bacteria or to prevent their growth.However, antibiotic resistance threatens the infective prevention andtreatment of an ever-increasing range of pathogenic bacteria. Antibioticresistance is present in all parts of the world. It is seen as anincreasingly serious threat to global public health that requires actionacross all sectors of science and society.

There are high proportions of antibiotic resistance in bacteria thatcause common infections in all regions of the world, e.g. urinary tractinfections, pneumonia, blood stream infections. A high percentage ofhospital-acquired infections are caused by so-calledmethicillin-resistant Staphylococcus aureus (MRSA). MRSA is a bacteriumresponsible for several difficult to-treat infections in humans. MRSA isalso called oxacillin-resistant Staphylococcus aureus (ORSA). MRSA isany strain of Staphylococcus aureus that has developed, through theprocess of natural selection, resistance to beta-lactam antibiotics,which include the penicillins, such as methicillin, dicloxacillin,nafcillin, oxacillin etc., as well as the cephalosporins. MRSA isespecially troublesome in hospitals, prisons, and nursing homes, wherepatients with open wounds, invasive devices, and weakened immune systemshave an elevated risk of a nosocomial infection in comparison to thegeneral public. MRSA began as a hospital-acquired infection, but hasdeveloped limited endemic status and is now increasinglycommunity-acquired.

Besides prevention measures new ways of treatment of MRSA are subject ofintensive research. According to a Henry Ford Hospital study the drug ofchoice for treating MRSA is now believed to be vancomycin. However,several newly discovered strains of MRSA show antibiotic resistance evento vancomycin, e.g. vancomycin-resistant Staphylococcus aureus (VRSA),as well as against other newly developed antibiotics intended for atreatment of MRSA.

Document DE 10 2005 055 944 discloses cyclic iminopeptide derivativesintended to be used as antibacterial agents. However, such knowncompounds have so far not proven their worth in the clinical practice.

SUMMARY OF THE INVENTION

Against this background it is an object underlying the invention toprovide a new anti infective compound effective in the treatment ofinfectious agents which are resistant to currently available drugs, suchas methicillin-resistant Staphylococcus aureus (MRSA),vancomycin-resistant Staphylococcus aureus (VRSA) orvancomycin-resistant Enterococci (VRE).

This object is met by the provision of a compound having the followingformula (I):

-   -   wherein    -   X is selected from the group consisting of: H, CH₃, CH₂CH₃,        anthranylalanine, DOPA, tyrosine, threonine,

-   -   under the proviso that at least one and, preferably at most two        or three, out of X is:

and

-   -   Y is selected from the group consisting of O, H, OH, S, and N,    -   under the proviso that in

-   -   -   ------------ represents no bond, single bond or double bond,        -   represents single bond or double bond,

    -   m is an integer between 0 and 3,

    -   n is an integer between 0 and 4,

    -   and the salts thereof, the solvates thereof and the solvates of        the salts thereof.

The inventors succeeded in isolating a cyclic peptide from the bacteriumStaphylococcus lugdunensis which falls under the scope of formula (I).This cyclic peptide exhibits strong activity against various pathogenicbacteria, including the vancomycin resistant (VRE) Enterococcus faecalisand E. faecium, Streptococcus pneumonia, Listeria monocytogenes, andStaphylococcus aureus. In further investigations the inventors were ableto identify a core structure represented by formula (I), which isresponsible for the antimicrobial activity of the isolated compound.

The new compound comprises 5-9 amino acids in a cyclic configuration. Inthis “large ring” among the amino acids aromatic and hydrophobic aminoacids are preferred, wherein at least one tryptophan or phenylalanine isrequired and in an embodiment preferably at most two, three, four etc.are tryptophan or phenylalanine. In an embodiment of the invention nomore than two aromatic amino acids are provided.

The new compound further may comprise a “small ring”, i.e. a 5-, 6- or7- membered heterocyclic ring, such as a a thiazolidine, oxazolidine, orimidazolidine ring. The small ring does not need a double bond, therecan be, however, a single or double bond in the small ring as indicatedby the dashed underlined line. Alternatively the “small ring” can beopened up without any ring structure as indicated by the dashed line.

The compound of the invention may, depending on its specific structure,exist in stereoisomeric forms (enantiomers, diastereomers). Theinvention therefore also encompasses the enantiomers or diastereomersand respective mixtures thereof. The stereoisomerically uniformconstituents can be isolated in a known manner from such mixtures ofenantiomers and/or diastereomers. If the compound of the invention mayoccur in tautomeric forms, the present invention encompasses alltautomeric forms.

Salts preferred for the purposes of the present invention arephysiologically acceptable salts of the compound of the invention. Alsoencompassed, however, are salts which are themselves not suitable forpharmaceutical applications but can be used for example for theisolation or purification of the compound of the invention.

Examples of pharmaceutically acceptable salts of the compound of formula(I) include salts of inorganic bases like ammonium salts, alkali metalsalts, in particular sodium or potassium salts, alkaline earth metalsalts, in particular magnesium or calcium salts; salts of organic bases,in particular salts derived from cyclohexylamine, benzylamine,octylamine, ethanolamine, diethanolamine, diethylamine, triethylamine,ethylenediamine, procaine, morpholine, pyrroline, piperidine,N-ethylpiperidine, N-methylmorpholine, piperazine as the organic base;or salts with basic amino acids, in particular lysine, arginine,ornithine and histidine.

Examples of pharmaceutically acceptable salts of the compound of formula(I) also include salts of inorganic acids like hydrochlorides,hydrobromides, sulfates, phosphates or phosphonates; salts of organicacids, in particular acetates, formates, propionates, lactates,citrates, fumarates, maleates, benzoates, tartrates, malates,methanesulfonates, ethanesulfonates, toluenesulfonates orbenzenesulfonates; or salts with acidic amino acids, in particularaspartate or glutamate.

Solvates for the purposes of the invention refer to those forms of thecompound of the invention, which in the solid or liquid state form acomplex by coordination with solvent molecules. Hydrates are a specificform of solvates in which the coordination takes place with water.

The compound of the invention may also be complexed, e.g. with iron,calcium, etc., in which the compound may act as a ligand, so that acorresponding complex is also subject of the present invention.

The problem underlying the invention is herewith completely solved.

According to another embodiment of the invention the compound ischaracterized by the following formula (II):

-   -   wherein    -   X is selected from the group consisting of: H, CH₃, CH₂CH₃,        anthranylalanine, DOPA, tyrosine, threonine,

-   -   under the proviso that at least one and, preferably at most two        or three, out of X is:

-   -   and the salts thereof, the solvates thereof and the solvates of        the salts thereof.

As the inventors were able to demonstrate, the compound according to theinvention consisting of a 7 amino acids comprising large ring and athiazolidin small ring has particularly strong antimicrobial activities.

The features, characteristics and advantages specified for the compoundrepresented by formula (I) apply to the compound represented by formula(II) as well.

According to another embodiment the compound according to the inventionis characterized by the following formula (III):

-   -   wherein    -   Y is selected from the group consisting of:

As the inventors were able to realize the compound according to theinvention represented by the core structure of formula (III) exhibits avery high antimicrobial activity.

The features, characteristics and advantages specified for the compoundrepresented by formulas (I) and (II) apply to the compound representedby formula (III) as well.

In another embodiment the compound according to the invention ischaracterized by the following formula (IV):

In this embodiment the exact compound is provided that was isolated bythe inventors from Staphylococcus lugdunensis. This compound has beenexemplarily used in the embodiments to demonstrate the antimicrobialactivity of compounds represented by the general formulas (I)-(III). Asthis has been demonstrated by the inventors, the compound hasbactericidal activity, therefore ensures an effective combating of abacterial infection. This compound can either be isolated fromStaphylococcus lugdunensis by well-known methods of microbiology incombination with chemical chromatography or synthesized by means ofpeptide synthesis.

The features, characteristics and advantages specified for the compoundrepresented by formulas (I), (II), and (III) apply to the compoundrepresented by formula (IV) as well.

In another embodiment of the invention the compound according to theinvention is provided for the treatment and/or prophylaxis of disease,preferably an infectious disease, further preferably a bacterialdisease, further preferably an infection by a Gram-positive bacterium,highly preferably an infection by Staphylococcus aureus, especiallyincluding its methicillin-resistant (MRSA) and vancomycin-resistantStaphylococcus aureus (VRSA) forms.

This measure has the advantage that a compound is provided beingeffective against a broad range of infectious agents and in particularagainst Staphylococcus aureus in its MRSA variant, which is responsiblefor several serious infections in humans.

Due to its pharmacological properties the compound of the invention canbe used alone or in combination with other agents for the treatmentand/or prophylaxis of diseases or infectious diseases, respectively, inparticularly bacterial infections.

For example, local and/or systemic diseases can be treated and/orprevented, which are caused by the following pathogens or by mixtures ofthe following pathogens:

Gram-positive cocci, such as Staphylococci (Staph. aureus, Staph.epidermidis) and Streptococcus (Strept. agalactiae, Enterococcusfaecalis, Strept. pneumonia, Strept. pyogenes), Gram-positive rods, suchas Bacillus (anthracis), Listeria (monocytogenes) and Corynebacterium(diphteriae), Gram-negative cocci (Neisseria gonorrhoeae) andGram-negative rods, such as Enterobacteriaceae, e.g. Escherichia coli,Haemophilus influenzae, Citrobacter (Citrob. freundii, Citrob.divernis), Salmonella and Shigella, further Klebsiella (Klebs.pneumoniae, Klebs. oxytoca), Enterobacter (Ent. aerogenes, Pantoeaagglomerans), Hafnia, Serratia (Serr. marcescens), Proteus (Pr.mirabilis, Pr. rettgeri, Pr. vulgaris), Providencia, Yersinia, and thegenus Acinetobacter. In addition, the antibacterial spectrum includesthe genus of Pseudomonas (Ps. aeruginosa and Ps. maltophilia) andstrictly anaerobic bacteria such as Bacteroides fragilis,representatives of the genus of Peptococcus, Peptostreptococcus and thegenus Clostridium; also mycoplasms (M. pneumoniae, M. hominis, M.urealyticum), and mycobacteria, for example Mycobacterium tuberculosis.

The above list of pathogens is merely to be illustrative and notlimiting.

Diseases caused by the pathogens mentioned or by mixed infections whichcan be cured, prevented, or attenuated by the compound according to theinvention are, for example:

Infectious diseases in humans such as septic infections, bone and jointinfections, skin infections, postoperative wound infections, abscesses,cellulitis, wound infections, infected burns, burns, infections of themouth, infections after dental surgery, septic arthritis, mastitis,tonsillitis, urogenital infections and eye infections.

Another embodiment of the invention relates to the use of the compoundaccording to the invention for the treatment and/or prophylaxis of nasalcolonization and infections.

According to the findings of the inventors herewith the compound is putinto place at the natural environment of action of the isolated cyclicpeptide.

Bacterial infections cannot only be treated or prevented in humans butalso in animals. Examples are:

Pig: coli diarrhea, enterotoxemia, sepsis, dysentery, salmonellosis,metritis-mastitis-agalactiae syndrome, mastitis;

Ruminants (cattle, sheep, goats): diarrhea, sepsis, bronchopneumonia,salmonellosis, pasteurellosis, mycoplasmosis and genital infections;

Horse: bronchopneumonia, puerperal and post puerperal infections,salmonellosis;

Dog and cat: bronchopneumonia, diarrhea, dermatitis, otitis, urinarytract infections, prostatitis;

Poultry (chicken, turkey, quail, pigeons, aviary birds and others):mycoplasmosis, E. coli infections, chronic airway disease,salmonellosis, pasteurellosis, psittacosis.

Similarly, bacterial diseases can be treated in the breeding and keepingof utility and ornamental fish but also humans, wherein theantibacterial spectrum of the pathogens mentioned above further extendsto pathogens such as Pasteurella, Brucella, Campylobacter, Listeria,Erysipelothris, Corynebacteria, Borrelia, Treponema, Nocardia,Rikettsia, Yersinia.

With the compound according to the invention bacterial infections cannotonly be treated or prevented in humans or animals but also plants.

The compound according to the invention can act systemically and/orlocally. For this purpose, it can be administered in a suitable manner,such as oral, parenteral, pulmonary, nasal, sublingual, lingual, buccal,rectal, dermal, transdermal, conjunctival, otic or associated with animplant or stent.

A topical application is especially preferred since the inventorssuccessfully demonstrated that the compound according to the inventionis able to penetrate even deeper tissue areas.

Against this background, another subject-matter of the invention is theuse of the compound according to the invention for the production of apharmaceutical composition for the treatment and/or prophylaxis of adisease, preferably an infectious disease, further preferably abacterial disease, further preferably an infection by a Gram-positivebacterium, highly preferably an infection by Staphylococcus aureus,including its methicillin-resistant form (MRSA).

Another subject-matter of the present invention is a pharmaceuticalcomposition comprising the compound according to the invention and apharmaceutical acceptable carrier.

For this purpose, a “pharmaceutically acceptable carrier” is understoodto mean any excipient, additive, or vehicle that is typically used inthe field of the treatment of infectious diseases and which simplifiesor enables the administration of the compound according to the inventionto a living being, and/or improves its stability and/or activity. Thepharmaceutical composition can also incorporate binding agents, dilutingagents or lubricants. The selection of a pharmaceutical carrier or otheradditives can be made on the basis of the intended administration routeand standard pharmaceutical practice. As pharmaceutical acceptablecarrier use can be made of solvents, extenders, or other liquid bindingmedia such as dispersing or suspending agents, surfactant, isotonicagents, spreaders or emulsifiers, preservatives, encapsulating agents,solid binding media, depending upon what is best suited for therespective dose regime and is likewise compatible with the compoundaccording to the invention. An overview of such additional ingredientscan be found in, for example, Rowe (Ed.) et al.: Handbook ofPharmaceutical Excipients, 7^(th) edition, 2012, Pharmaceutical Press.

According to another embodiment of the invention the pharmaceuticalcomposition is provided for the treatment and/or prophylaxis of adisease, preferably an infectious disease, further preferably abacterial disease, further preferably an infection by a Gram-positivebacterium, highly preferably an infection by Staphylococcus aureusincluding its methicillin resistant forms (MRSA).

The features, characteristics, advantages and embodiments of thecompound according to the invention likewise apply to the use and thepharmaceutical composition according to the invention.

Another subject-matter of the present invention is a method of producingthe compound according to the invention, comprising the followingsteps: 1. Providing bacteria of the species of Staphylococcuslugdunensis, 2. Purifying the compound according to the invention fromsaid bacteria.

Herewith a method is provided, which has been used by the inventors forproducing or isolating the compound according to the invention. Thespecies of Staphylococcus lugdunensis referred to in this embodiments orelsewhere in the disclosure includes the isolate or strain IVK28,respectively.

In an embodiment of the method according to the invention after step (1)and before step (2) the bacteria and the medium of the bacterial cultureare extracted and the bacterial and the medium extract are subjected tostep (2) where the compound is purified from said bacterial and mediumextract.

This measure has the advantage that only the main sources of thecompound according to the invention are provided to the purifying stepfrom which it can then be isolated by methods of microbiology incombination with chemical chromatography, which are well known to theskilled person.

In another embodiment of the method according to the invention in step(2) the purification involves the use of high performance liquidchromatography (HPLC) to identify a peak signal, which is associatedwith said compound.

This measure has the advantage that a well-established tool of peptidepurification is used, which allows a reliable identification of thecompound according to the invention.

In another embodiment of the method according to the invention saidsignal peak corresponds to a molecular mass of approx. 650 Da-950 Da,preferably of approx. 700 Da-850 Da, further preferably of approx. 750Da-800 Da, further preferably of approx. 770 Da-790 Da, and highlypreferably of approx. 782.5 Da.

This measure has the advantage that the compound according to theinvention, which falls within the indicated ranges, can be easilyidentified via its molecular mass.

Another subject-matter of the present invention is a method of producingthe compound according to the invention, which comprises the followingsteps: 1. Expressing the non-ribosomal peptide synthetase system II ofthe species of Staphylococcus lugdunensis (NRPS-II) in a biologicalsystem, 2. Incubating the expressed NRPS-II under conditions allowingthe synthesis of the compound according to the invention, 3. Purifyingsaid compound.

The inventors were able to find out that the NRPS-II of the species ofStaphylococcus lugdunensis is responsible for the synthesis of thecompound according to the invention and, therefore, this method makesuse of the natural apparatus for producing the new compound.

In an embodiment of the method according to the invention said NRPS-IIis encoded by a nucleic acid molecule comprising any of the codingsequences of the genes lugA, lugB, lugC, lugD, and, optionally, of atranscriptional regulator of GntR family, ABC transporter (GdmF typemultidrug transport system), ABC-2 type transport system permeaseprotein, ABC transporter, hyp. membrane protein, TetR/AcrR familyregulator, thioesterase family protein,4′-phosphopantetheinyltransferase, and put. negative regulator of sigY(in Bacillus).

The inventors were able to identify the genes responsible for thesynthesis of the compound according to the invention and the optionalco-factors, and, therefore, provide a method where the essentialfeatures are used in a molecular biological artificial system togenerate the compounds in a large scale.

The coding sequences comprised by the NRPS-II can be taken from SEQ IDNO: 1; the nucleotide positions are indicated in the following:

-   -   lugA: 6007-13131    -   lugB: 13121-17341    -   lugC: 17359-26172    -   lugD: 26893-28632    -   transcriptional regulator of GntR family: 427-936    -   ABC transporter (GdmF type multidrug transport system):        1253-2143    -   ABC-2 type transport system permease protein: 2140-2904    -   ABC transporter: 2917-3630    -   hyp. membrane protein: 3623-5164    -   TetR/AcrR family regulator: 5409-5981    -   Thioesterase family protein: 26169-26855    -   4′-phosphopantetheinyltransferase: 28640-29293    -   put. negative regulator of sigY (in Bacillus): 1027-1266

Against this background, another subject-matter of the present inventionis a nucleic acid molecule comprising the nucleotide sequence of SEQ IDNO: 1 and/or any of the before-listed coding sequences of thenon-ribosomal peptide synthetase system II of the species ofStaphylococcus lugdunensis (NRPS-II). Examples for such nucleic acidmolecules include vectors or plasmids configured for a controlledexpression of the encoded proteins in common molecular biologicalexpression systems. Subject of the invention is also a nucleic acidmolecule encoding the identical protein and/or peptide as encoded by thebefore mentioned nucleic acid molecule, however having a modified ordifferent nucleotide sequence due to the degeneration of the geneticcode.

Another subject-matter of the invention relates to the productsgenerated by the expression of the nucleic acid molecule according tothe invention, including proteins and/or peptides encoded by any of thegenes lugA, lugB, lugC, lugD, transcriptional regulator of GntR family,ABC transporter (GdmF type multidrug transport system), ABC-2 typetransport system permease protein, ABC transporter, hyp. membraneprotein, TetR/AcrR family regulator, thioesterase family protein,4′-phosphopantetheinyl-transferase, and put. negative regulator of sigY(in Bacillus), or encoded by before mentioned nucleotide sequence whichare itslef subject-matter of the present invention.

A further subject-matter of the invention relates to an antibodyspecifically directed against any of the products (peptides, proteins)generated by the expression of the nucleic acid molecules according tothe invention.

In another embodiment of the method according to the invention theconditions allowing the synthesis of the compound according to theinvention comprise amino acids, thioesterase, and buffer.

By this measure the conditions are adjusted in a way, which ensures ahigh yield of the compound according to the invention.

Another object of the present invention is a method for the treatmentand/or prophylaxis of diseases, especially of the aforementioneddiseases, in a living being, such a human or animal being, comprisingthe administration to said living being of an antibacterially effectiveamount of the compound according to the invention.

Another object of the invention is a probiotic comprising amicororganism capable of producing the compound according to theinvention.

The inventors have surprisingly realized that a micororganism, such as abacterium, producing the novel anti-bacterial compound can be directlyused, e.g. to prevent or reduce the growing or colonization by apathogenic microorganism of an organ of a living being.

According to the invention, a “probiotic” is to be understood as amicroorganism which provides health benefits to a living being, such asa human being, when consumed or administered. In particular, theadministration of the probiotic to an organ susceptible of beingcolonized by a pathogen microorganism, such as Staphylococcus aureus,will result in a displacement of such pathogenic agent. This results ina reduction of the pathogenic burden or even in a complete elimination.

In another embodiment the microorganism is Staphylococcus lugdunensis.Staphylococcus lugdunensis can be used in its natural form, i.e. as thewild type, or as a modified form, e.g. a genetically modified form or anattenuated wild type variant, however still producing the compoundaccording to the invention.

In another embodiment of the invention the probiotic is used forpreventing or reducing the colonization by a pathogenic microorganism ofan organ of a living being, e.g. a human being. The organ may be thenose, wherein the probiotic may then be administered into the nose ofthe living being, or it may be the skin, wherein the probiotic may thenbe administered onto the skin of the living being.

It is to be understood that the before-mentioned features and those tobe mentioned in the following cannot only be used in the combinationindicated in the respective case, but also in other combinations or inan isolated manner without departing from the scope of the invention.

The invention is now further explained by means of embodiments resultingin additional features, characteristics and advantages of the invention.The embodiments are of pure illustrative nature and do not limit thescope or range of the invention.

The features mentioned in the specific embodiments are also features ofthe invention in general, which are not only applicable in therespective embodiment but also in an isolated manner in the context ofany embodiment of the invention.

The invention is also described and explained in further detail byreferring to the following drawings

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the lugdunin production by S. lugdunensis wild type andisogenic mutants. Bioactivity assay with the S. lugdunensis IVK28 wildtype, the ΔlugD mutant and the complemented mutant against S. aureusUSA300. BM plates were inoculated with a lawn of S. aureus USA300.Hereon S. lugdunensis IVK28 cells from overnight cultures of the wildtype, the mutant ΔlugD and the complemented mutant ΔlugD::pRB474/lugDwere spotted.

FIG. 2 shows the genetic locus required for the synthesis of lugduninand the domain architecture of the S. lugdunensis IVK28 non-ribosomalpeptide synthetases with predicted specificities;

FIGS. 3A and 3B show the identification of the NRPS-II product in cellextracts of S. lugdunensis IVK28. FIG. 3A shows HPLC-UV chromatogram(UV_(210 nm)) of cell extracts of S. lugdunensis wild type (blue) andmutant M1 (red) (x-axis: time [min], y-axis adsorption at 210 nm [mAU];FIG. 3B shows HPLC-MS of the peak at 10.6 min retention time resultingin a mass of 782.5 Da (ESI pos.: m/z 783.6 [M+H]⁺, ESI neg.: m/z 781.5[M-H]⁻, m/z 817.5 [M+Cl]⁻);

FIGS. 4A to 4C show that S. lugdunensis IVK28 is able to eliminate S.aureus USA300. S. aureus USA300 (black) and S. lugdunensis IVK28 wildtype (FIG. 4A) or mutant ΔlugD (FIG. 4B and FIG. 4C) (white) were mixedin varying ratios (10:1 (FIG. 4A and FIG. 4B) or 1:10 (FIG. 4C)) andspotted on BM-agar containing 2,2′-bipyridine. After the indicated timepoints the ratio of the strains in the spot was determined. The figuresrepresent the mean of three experiments;

FIG. 5A shows the HPLC-MS-MS fragments and FIG. 5B shows the structuralformula of the cyclic peptide embodying the compound according to theinvention (“lugdunin”);

FIGS. 6A and 6B show that lugdunin has bactericidal activity and notendency to induce spontaneous resistance. FIG. 6A shows a killingcurve. Incubation of S. aureus with a 10×MIC of lugdunin leads tocomplete killing of the inoculum after 30 h. Data represent medians±S.D. of three independent experiments. FIG. 6B shows serial passagingof S. aureus with sub-inhibitory concentrations of rifampicin leads torapidly increasing spontaneous resistance against rifampicin. However,such resistance development was not observed with lugdunin. Arepresentative of two independent experiments is shown.

FIG. 7: shows that lugdunin exhibits bactericidal activity againstnon-growing S. aureus USA300. Incubation of S. aureus USA300 with 1.5μg/ml lugdunin leads to an at least 100-fold decrease of viable cells inPBS within 6 hours;

FIG. 8: shows that lugdunin is active in vivo in a mouse tape-strippingmodel. After colonization of shaved C57BL/6 mouse skin with S. aureusNewman lugdunin was applied three times in a few hours distance. After45 hours mice were sacrificed and the numbers of bacteria weredetermined on the skin surface (wash fraction) and in the deeper skintissue (scrape fraction). Treatment with lugdunin significantly reducedthe bacterial load in both fractions indicating its efficacy also invivo. (*p<0.05);

FIG. 9: shows a cytotoxicity test or the determination of potentialcytotoxic effects on neutrophil granulocytes within 3 h incubation.

FIGS. 10A to 10E show that Lugdunin-producing S. lugdunensis eradicatesS. aureus in vitro and in vivo in cotton rats. FIG. 10A shows S. aureusis overgrown by S. lugdunensis IVK28 wild type on agar plates inoculatedat ratios of ˜90:10. In contrast, FIG. 10B shows IVK28 ΔlugD isovergrown by S. aureus at ratios of ˜90:10 (b). FIG. 10C shows thecapacity of S. lugdunensis ΔlugD to overgrow S. aureus is largelyrestored by plasmid-encoded lugD. FIG. 10D shows S. aureus overgrowsIVK28 ΔlugD even when inoculated in ratios of ˜10:90. Data representmean values ±S.D. of three independent experiments. Significantdifferences between starting conditions and indicated time points wereanalysed by one-way ANOVA (*, p<0.05; **, p<0.01; ***, p<0.001; ****,p<0.0001; n.s, not significant). FIG. 10E shows cotton rat nosesco-colonised by S. aureus and S. lugdunensis IVK28 wild type showsignificantly less S. aureus CFUs after five days compared to S.lugdunensis IVK28 ΔlugD. Horizontal lines represent the median of eachgroup. Significant differences, calculated by the Mann Whitney test, areindicated (*, p<0.05).

FIGS. 11A to 11C show the nasal colonization rates in cotton rats of S.aureus and S. lugdunensis. Different inocula of S. aureus Newman (FIG.11A), S. lugdunensis IVK 28 wild type (FIG. 11 B) and S. lugdunensis IVK28 ΔlugD (FIG. 11C) were instilled intranasally to determine theircolonization efficiency in cotton rats. CFUs of each strain weredetermined per nose after 5 days and plotted as individual dots in FIGS.11A-11C. Lines represent the median of each group.

FIG. 12: shows the distribution of carriage of S. aureus, S.lugdunensis, and both in a sample of 187 risk patients.

DESCRIPTION OF PREFERRED EMBODIMENTS

A. Methods

1. Strains and Growth Conditions

The Staphylococcus strains used in this study were S. aureus USA300 LAC,S. aureus USA300 NRS384, S. aureus Mu50, S. aureus RN4220, S. aureusSA113, S. aureus Newman, S. aureus PS187, S. lugdunensis IVK28, S.lugdunensis IVK28 ΔlugD, S. lugdunensis IVK28 ΔlugD::pRB474/lugD, and S.lugdunensis IVK28-Xyl. Further strains used for MIC determination wereEnterococcus faecium BK463, E. faecalis VRE366, Listeria monocytogenesATCC19118, Streptococcus pneumoniae ATCC49619, Pseudomonas aeruginosaPAO1, and Escherichia coli DH5α. E. coli DC10B was used as the cloninghost. In addition, a set of 60 S. aureus and 17 S. lugdunensis strainswere isolated from diagnostic samples in the course of the colonisationstudy described below.

Basic medium (BM: 1% soy peptone, 0.5% yeast extract, 0.5% NaCl, 0.1%glucose and 0.1% K₂HPO₄, pH 7.2) was used as the standard growth medium.MIC determinations and killing assays were performed in Mueller HintonBroth (MHB; Roth, Karlsruhe, Germany). For the identification of S.lugdunensis, selective S. lugdunensis medium (SSL) was used aspreviously described in the art. When necessary, antibiotics were usedat concentrations of 250 μg/mL for streptomycin, 10 μg/mL forchloramphenicol, 2.5 μg/mL for erythromycin and 100 μg/mL forampicillin.

2. Bioactivity Test

The anti-S. aureus activity of S. lugdunensis IVK28 was identified byscreening 90 nasal staphylococcal isolates for the capacity to inhibitgrowth of S. aureus. For this purpose BM agar was inoculated 1:10,000with an overnight culture of S. aureus USA300 LAC. The test strains wereinoculated on the resulting bacterial lawn, and the plates wereincubated for 24-48 h at 37° C. To investigate the production ofantimicrobial activity by IVK28 under iron-limiting conditions, BM agarwas supplemented with 200 μM 2,2′-bipyridine.

3. Transposon Mutagenesis and Elucidation of the Lugdunin Gene Cluster

The temperature-sensitive plasmid pTV1ts, which contains the 5.3-kbtransposon Tn917 (erm^(R)) from E. faecalis, was transferred into S.lugdunensis IVK28 by electroporation. Transposition mutants werescreened for loss of antimicrobial activity against S. aureus.Chromosomal DNA was isolated by standard procedures from non-inhibitoryclones, and the primers Tn917 up and Tn917 down (Extended Table 2) wereused to directly sequence the flanking regions of the transposoninsertion site. Sequence analysis was performed with DNASTAR Lasergenesoftware (DNASTAR Inc., Madison, Wis., USA). Bioinformatic analysis wasperformed by BLAST® and antiSMASH 3.0.

4. Generation of S. lugdunensis IVK28-Xyl

The flanking regions of lugR were amplified by PCR with the primer pairsSIPr1-up/SIPr1-down and SIPr2-up/SIPr2-down. The plasmid pBASE6-erm/lox1, a derivative of pBASE6, already containing an erythromycin resistancecassette in the singular SmaI site, was linearized with Acc65I. Theidentically digested SIPr1 PCR product, containing one natural Acc65Irestriction site and one introduced by the primer, was ligated intopBASE6-erm/lox1.The resulting vector with the correctly oriented SIPr1PCR product and the SIPr2 PCR product were ligated after digestion withEcoRV and BgIII. The resulting pBASE6-erm/lox 1 construct with bothflanking regions inserted was linearized with BssHII, treated withKlenow enzyme and digested with BgIII. The required xylR fragment withthe downstream-located xylAB-promoter was excised from pTX15 by HindIIIrestriction treatment with Klenow enzyme and subsequent digestion withBamHI. The ligation of the xylR fragment into the appropriate vectorgenerated pBASE6-erm/lox1-xylR, which was transferred into E. coli DC10Band subsequently into S. aureus PS187. The resulting plasmidpBASE6-erm/lox1-xylR was transduced into S. lugdunensis IVK28 via thebacteriophage Φ187. Homologous recombination for replacement of lugR byerm/xylR was performed, as previously described in the art, generatingthe xylose-inducible lugdunin producer strain S. lugdunensis IVK28-Xyl.

5. Production and Purification of Lugdunin

A fresh overnight culture of S. lugdunensis IVK28-Xyl was inoculated1:1,000 in BM without glucose and was supplemented with 0.5% xylose.After incubation at 37° C. under continuous shaking (160 rpm) for 24 h,whole cultures were extracted with 1-butanol at a ratio of 5:1. Theaqueous phase was discarded, and the organic phase was evaporated at 37°C. under reduced pressure and finally dissolved in methanol. Themethanol extract was applied to a gel filtration column (Sephadex LH20,1.6×80 cm, flow rate 1 mL/min). The active fractions containing lugduninwere pooled, evaporated at 37° C. under reduced pressure and dissolvedin dimethyl sulfoxide (DMSO). This solution was then subjected to apreparative reverse-phase HPLC column (Kromasil C18, 7 μm, 250×20 mm;Dr. Maisch, Ammerbuch, Germany) with an isocratic elution at 79%methanol in water for 20 min. The peak containing lugdunin wasbaseline-separated from the remaining compounds, and methanol wasevaporated at 37° C. under reduced pressure to yield a white powder ofpure lugdunin.

6. Chemical Synthesis of Lugdunin and Lugdunin Derivatives

Total chemical synthesis was achieved by Fmoc(9-fluorenylmethoxycarbonyl) strategy based manual solid-phase peptidesynthesis and was established on an H-Val-H NovaSyn® TG resin(Novabiochem, Switzerland). Amino acids were coupled in a four-foldexcess using HATU(1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate). Valine positions were coupled twice by useof PyOxim ([Ethylcyano(hydroxyimino)acetato-O²]tri-1-pyrrolidinylphosphoniumhexafluorophosphate) for the second coupling instead of HATU. Peptideswere cleaved from the resin with acetonitrile/water/trifluoroacetic acid(79.95/20/0.05) for 30 min. Lyophylization overnight yielded the crudeproduct. Crude synthetic lugdunin products were purified by RP-HPLC andcompared with the natural product by HR-LC-ESI-MS, additionalchiral-HPLC methods (column: Dr. Maisch Reprosil Chiral NR, Ammerbuch,Germany; elution with 80% premixed methanol in H₂O at 1.5 mL/min flowrate), bioactivity assay and advanced Marfey's analysis.

7. MIC Assay and Spectrum of Activity

S. aureus RN4220, S. aureus USA300 (LAC), S. aureus USA300 (NRS384), S.aureus SA113, S. aureus Mu50, E. coli DH5α and P. aeruginosa PAO1 weregrown overnight in MHB. E. faecalis VRE366, E. faecium BK463, S.pneumoniae, and L. monocytogenes were grown in tryptic soy broth (TSB:Difco Laboratories, Augsburg, Germany). All strains were incubated at37° C. under continuous shaking. Early log-phase grown bacteria wereadjusted in MHB to 1×10⁶ cells/well in microtiter plates (MTP), mixedwith varying concentrations of the antibiotic and incubated at 37° C.for 24 h under continuous shaking. The OD₆₀₀ of each well was measuredwith a microplate reader, and the lowest peptide concentrations, whichdisplayed no bacterial growth, were defined as the MIC. The assays wereperformed in 96-well microtiter plates.

8. Killing Assay

Fresh MHB was inoculated 1:10,000 with an overnight culture of S. aureusUSA300 LAC and was incubated at 37° C. under continuous shaking (160rpm) until bacteria were grown to 1×10⁶ cells/mL. Then, 10×MIC lugduninwas added. At the time points 0 h, 2 h, 4 h, 8 h, 24 h and 30 h, sampleswere taken and centrifuged. The pellet was resuspended in 1×PBS andserially diluted. The dilutions were spotted on tryptic soy agar, andcolony counts were determined after overnight incubation at 37° C. Todetermine cell numbers <10² cells/mL, whole cultures of 1 mL werecentrifuged and plated on TSA.

9. Cytotoxity Against Human Neutrophil Granulocytes

Human neutrophil granulocytes were freshly isolated from the blood ofhealthy volunteers by standard Histopaque/Ficoll centrifugation. Thelysis of neutrophil granulocytes was monitored by the release of theenzyme lactate dehydrogenase (LDH). Lugdunin was added at finalconcentrations of 50, 25 and 12.5 μg/mL in 0.5% DMSO to wells of a 96well tissue culturing plate containing 1×10⁶ neutrophil granulocytes perwell in 200 μL RPMI-1640 medium (2 g/l NaHCO₃, 10% foetal calf serum, 1%L-glutamine and 1% penicillin-streptomycin, PAN Biotech) without phenolred. The plates were incubated at 37° C. and 5% CO₂ for 3 h and thelysis was determined with a Cytotoxicity Detection Kit (Roche AppliedSciences, Mannheim, Germany). As a positive control for highcytotoxicity, 2% Triton X-100 was added to the samples.

10. Resistance Development Study

MIC assays for the antibiotics used in this study were performed asdescribed above. The inventors determined 1×MICs of 0.01 μg/mLrifampicin and 1.5 μg/mL lugdunin against S. aureus USA300. Fresh MHBwas inoculated 1:10,000 with an overnight culture of S. aureus USA300LAC and was incubated at 37° C. under continuous shaking. Cells weregrown to early log phase, adjusted to 1×10⁶ cells/mL, and dispensed into96-well MTPs with 100 μL per well. Lugdunin and rifampicin were added atconcentrations of 0.25×MIC, 0.5×MIC, 1×MIC, 1.5×MIC, 2×MIC and 4×MIC.After 24 h incubation at 37° C. under continuous shaking, growth wasdetermined with a microplate reader at an OD₆₀₀ and cells from thesecond highest concentration of 0.25×MIC, 0.5×MIC, 1×MIC, 1.5×MIC, 2×MICand 4×MIC from the appropriate antibiotic.

11. Statistical Analyses

Statistical analysis was performed by using GraphPad Prism (GraphPadSoftware, Inc., La Jolla, USA; version 5.04). Statistically significantdifferences were calculated by using appropriate statistical methods asindicated. For the human study, risk of nasal colonisation with S.aureus under the presence and absence of S. lugdunensis, as well as therespective point estimates of the risk ratio and confidence intervals,were determined using Stata version 12.0 (Stat Corp., College Station,Tex., USA). P values of ≤0.05 were considered significant.

12. Animal Models and Ethics Statement

All animal experiments were conducted in strict accordance with theGerman regulations of the Gesellschaft für Versuchstierkunde/Society forLaboratory Animal Science (GV-SOLAS) and the European Health Law of theFederation of Laboratory Animal Science Associations (FELASA) inaccordance with German laws after approval (protocol HT1/12 for mouseskin infection and T1/10 for cotton rat colonisation) by the localauthorities (Regierungspraesidium Tuebingen). All, animal and humanstudies, were carried out at the University Hospital, Tuebingen, andconformed to institutional animal care and use policies. Norandomization or blinding was necessary for the animalinfection/colonisation models, and no samples were excluded. Animalstudies were performed with female C57BL/6 mice, 6-8 weeks old andcotton rats of both genders, 8-10 weeks old, respectively. The humannasal colonization study was approved by the ethics committee of themedical faculty of the University Hospital Tuebingen (project number577/2015A).

13. Skin Infection of C57BL/6 Mice

A streptomycin-resistant S. aureus Newman strain was used to infectC57BL/6 mice epicutaneously by the tape stripping technique. TSB with500 μg/mL streptomycin was inoculated 1:10,000 with a fresh overnightculture of the test strain and was incubated at 37° C. under continuousshaking until an OD₆₀₀=0.5 was reached. Cells were harvested, washedtwice with 1×PBS, and adjusted to 1×10⁸ cells/mL. The integrity of theshaved skin of the mice was affected by repeated (seven times) vigoroustape stripping to enable S. aureus Newman infection. An inoculum of 15μL from the bacterial suspension was added to 7-mm filter paper discs,placed onto the prepared skin with two discs per animal, and coveredwith Finn chambers on Scanpor tape (Smart Practise, Phoenix, Ariz.,USA). Finn chamber fixation occurred via Fixomull stretch plasters (BSNmedical GmbH, Hamburg, Germany). After incubation for 24 h, the Finnchambers were removed and 1.5 μg of lugdunin per colonised area wasapplied, followed by a second and third treatment with the same amountof lugdunin after 30 h and 42 h. Six hours after the final application,mice were euthanized, the skin was large-scale detached and 4-mm punchesof the originally colonised areas were vortexed in 1×PBS for 30 secondsto remove the attached bacteria from the skin (wash fraction). The skinwas dissected with a scalpel to expose bacteria from deeper areas of theskin (tissue fraction), which was homogenized by vortexing in 1×PBS for30 seconds. CFUs of both fractions were determined by serial dilutionsin 1×PBS, which were then spotted onto TSA, supplemented withstreptomycin, for S. aureus Newman^(strep) specific selection. Theplates were incubated overnight at 37° C.

14. Generation of S. lugdunensis ΔlugD and Complementation

For the construction of a marker-less knock-out strain, 1-kb flankingregions of lugD were amplified by PCR with the primer pairs lugDupstream-SacI/lugD upstream-Acc65I and lugD downstream-Acc65I/lugDdownstream-BgIII. The fragments were digested according to theirintroduced restriction sites and were ligated into the plasmid pBASE6generating pBASE6-ΔlugD, which was transferred into E. coli DC10B. Thecorrect plasmid was transferred to S. aureus PS187 by electroporation,which was then infected with the bacteriophage Φ187 for the transductionof pBASE6-ΔlugD into the S. lugdunensis IVK28 wild type. The knockoutwas generated by homologous recombination of the flanking regions intothe genome, and deletion of lugD was confirmed by PCR. For thecomplementation of the mutant, lugD was amplified by the primer pairlugD comp. forw-PstI/lugD comp. rev-Acc65l, digested with theappropriate restriction enzymes and ligated into identically digestedpRB474. The constructed pRB474-lugD was transduced into S. lugdunensisIVK28 ΔlugD, as described for the knock-out mutant.

15. Competition Assay

S. lugdunensis IVK28 wild type, S. lugdunensis IVK28 ΔlugD, S.lugdunensis IVK28 ΔlugD::pRB474-lugD, and a streptomycin-resistant S.aureus Newman were grown in BM overnight at 37° C. under continuousshaking. These strains were then adjusted to 1×10⁹ cells/mL in 1×PBS anddiluted 1:10. For the starting condition of 90% S. aureus, equal volumesof 1×10⁹ S. aureus cells/mL and 1×10⁸ S. lugdunensis cells/mL weremixed. Co-cultures with only 10% S. aureus were also performed, and 20μL of these mixtures were spotted in triplicate on BM agar and incubatedat 37° C. Samples were taken at 0 h, 24 h, 48 h and 72 h by scrapingcells from the agar plates and suspending them in 1×PBS. Serialdilutions of these samples were plated on BM and BM containingstreptomycin for selection of S. aureus. After overnight incubation at37° C., colony counts were determined, and the bacterial ratios of S.aureus and S. lugdunensis were calculated.

16. Co-Colonisation of Cotton Rat Noses

For the colonisation of cotton rat noses, spontaneousstreptomycin-resistant mutants of S. lugdunensis IVK28 wild type and S.lugdunensis IVK28 ΔlugD were selected on BM agar plates containing 250μg/mL streptomycin. Co-colonisation was conducted with S. aureusNewman^(strep). The cotton rat model was described earlier. Since thecapacity of S. lugdunensis to colonize cotton rat nares has not beenstudied before, the inventors determined the inoculum required forstable colonization by IVK28 wild type and its mutant ΔlugD over 5 days.The inventors' previous studies have shown that for S. aureus aninoculum of 10⁷ bacteria per nose results in a constant colonization ofabout 10³ CFUs per nose. To achieve a comparable colonization level withS. lugdunensis, an inoculum of 10⁸ bacteria per nose was required, andthere was no detectable difference in colonization efficiency betweenwild type and ΔlugD. Therefore, co-colonization experiments in cottonrat noses were performed with 10-fold more S. lugdunensis than S. aureusto obtain a 1:1 colonization ratio.

Cotton rats were anesthetized and instilled intranasally with mixturesof either 1×10⁸ S. lugdunensis wild type/1×10⁷ S. aureus Newman or 1×10⁸S. lugdunensis ΔlugD/1×10⁷ S. aureus Newman. Five days after bacterialinstillation, the animals were euthanized, and noses were surgicallyremoved. The noses were heavily vortexed in 1 mL of 1×PBS for 30 s.Dilutions of the samples in PBS were plated on SSL agar containing 250μg/mL streptomycin to select for the used strains and to separate S.aureus (yellow) and S. lugdunensis (purple) by colour. The plates wereincubated for two days under anaerobic conditions (anaerobic jar withAnaerocult® A, MerckKGaA), for the specific detection of ornithinedecarboxylase activity. S. aureus Newman CFUs were determinedafterwards. All animals received drinking water with 2.5 mg/mLstreptomycin continuously, starting three days prior to the experiment,to reduce the natural nasal flora.

17. Human Colonisation Study

A total of 187 nasal swab samples from hospitalized patients werereceived from the diagnostics laboratory of the Institute of MedicalMicrobiology and Hygiene (University Clinic Tuebingen, Germany).Dilutions from each sample were plated on blood agar and SSL agar for aphenotypic identification of S. aureus and S. lugdunensis. Identity wasconfirmed by coagulase test and matrix-assisted laserdesorption/ionization-time-of-flight mass spectrometry (Massspectrometer: AXIMA Assurance, Shimadzu Europa GmbH, Duisburg, Database:SARAMIS™ with 23.980 spectra and 3.380 super-spectra, BioMérieux,Nuertingen).

B. Results

1. Staphylococcus lugdunensis Produces a Highly Potent AntimicrobialCyclic NRPS-Peptide with Strong Activity Against Staphylococcus aureus

In natural habitats, especially in nutrient-poor ecological niches likethe human nose [Krismer et al. (2014) Nutrient limitation governsStaphylococcus aureus metabolism and niche adaptation in the human nose.PLoS Pathog 10: e1003862], a fierce competition about availablenutrients between colonizing bacteria is assumed. The inventors screenedbacterial isolates from nasal swabs for the production of compoundsactive against S. aureus. Beside activities against a huge range ofvarious nasal bacteria the inventors identified two strains withinhibiting properties against S. aureus. Whereas one isolate, which wasidentified as Staphylococcus epidermidis, exhibited constant productionof the antibacterial activity under the investigated conditions, thesecond isolate, Staphylococcus lugdunensis IVK28 was found to have aparticularly strong capacity to prevent the growth of S. aureus (FIG.1). IVK28 showed its antibacterial effect only under iron-limitingconditions. No such inhibitory activity has been described yet forstaphylococci. For this reason, this IVK28 designated isolate of S.lugdunensis was further investigated by the inventors.

2. The Genetic Organization of the S. lugdunensis IVK28 NRPS-Operon

Subsequent transposon mutagenesis of the strain resulted in theproduction-negative mutant M1. Analysis of the insertion site by inversePCR resulted in the identification of a gene encoding a non-ribosomalpeptide synthetase (NRPS; position 860375/76 in the annotated genomesequence of S. lugdunensis N920143; Acc.no: FR870271.1) which is part ofthe NRPS-II designated system. This clearly indicated that a smallpeptide might exhibit the antibacterial activity of S. lugdunensisagainst S. aureus. In the S. lugdunensis genome three putativeNRPS-systems have been identified [The entire genome sequence ofStaphylococcus lugdunensis N920143 is published in Heilbronner, et al.(2011) Genome sequence of Staphylococcus lugdunensis N920143 allowsidentification of putative colonization and virulence factors. FEMSMicrobiol Lett 322: 60-67, which is incorporated herein by reference].Whereas NRPS-I exhibits high homologies to the S. aureus NRPS dipeptidesystem encoding aureusimine A and B [Wyatt et al. (2010) Staphylococcusaureus nonribosomal peptide secondary metabolites regulate virulence.Science 329: 294-296] [Erratum in Science, 2011 Sep. 9; 333(6048):1381], and NRPS-III has striking similarities to describedsiderophore systems (Heilbronner et al., cit loc.) nothing is knownabout the potential product encoded by NRPS-II. Investigation of thepublished genomes of S. lugdunensis (completed sequences of strainsN920143, HKU09-01 and partial sequences of strains VCU139 and M235909(can easily determined on the basis of the genome sequence ofStaphylococcus lugdunensis N920143. HKU09-01: between 864800/864801)showed that the applying NRPS-II operon is present in every strainsequenced so far and therefore does not depict a strain specificfeature. Nevertheless, the published genomes do contain variouspotential sequencing errors or real frame-shift mutations, leading todifferent annotations. For this reason, all relevant positions,indicating a potential frame-shift, were amplified by PCR from strainIVK28. Subsequent sequencing of the PCR products revealed that thesequence of IVK28 corresponds to that of strain N920143 and itsannotation, except for the annotated nucleotide position 863515 which islocated at the 3′-end of gene SLUG_08110. In contrast to N920143, inIVK28 there is a stretch of eight instead of sevenadenosine-nucleotides, leading to the fusion of genes SLUG_08110 andSLUG_08120 to one open reading frame.

FIG. 2 depicts the genetic organization of the 29.6 kb NRPS-II operon inS. lugdunensis IVK28. The locations of the coding sequences areindicated in the following sequence information of the S. lugdunensisIVK28 NRPS-operon:

LOCUS nrp-operon IVK28 29605 bp DNA linear FEATURES  Location/Qualifiersmisc_feature  complement (427..936)  /note=“transcriptional regulator ofGntR family.” misc_feature  1253..2143  /note=“ABC transporter (GdmFtype multidrug  transport  system)” misc_feature  2140..2904 /note=“ABC-2 type transport system permease  protein” misc_feature 2917..3630  /note=“ABC transporter” misc_feature  3623..5164 /note=“hyp. membrane protein” misc_feature  5409..5981 /note=“TetR/AcrR family regulator” misc_feature  6007..13131/note=“lugA” (corresponds to SLUG_08100 of Staphylococcus lugdunensisN920143) misc_feature  13121..17341 /note=“lugB” corresponds to thefused sequence of SLUG_08110 and SLUG_08120 (by insertion of oneadditional nu- cleotide) of Staphylococcus lugdunensis N920143)misc_feature  17359..26172 /note=“lugC” corresponds to SLUG_08130 ofStaphylococcus lugdunensis N920143) misc_feature  26169..26855 /note=“Thioesterase family protein” misc_feature  26893..28632/note=“lugD” corresponds to SLUG_08150 of Staphylococcus lugdunensisN920143) misc_feature  28640..29293 /note=“4′-phosphopantetheinyltransferase” misc_feature  1027..1266/note=“put. negative regulator of sigY (in Bacillus” misc_feature 10338..10347  /note=“Tn917 insertion site” BASE COUNT 11616 a 3273c 4650 g 10066 t

Interestingly, with only 26.7% the GC-content of the NRPS-operon issignificantly lower than the overall GC-content of the genome (33.8%),indicating horizontal gene transfer from an extremely low GC-organism.The operon contains four consecutive genes (named lugA, B, C, D)encoding NRPS proteins, interrupted by a type II thioesterase genebetween lugC and lugD. In the 5′-region two ABC-transporters and twopotential regulatory genes are encoded. At the 3′-end of the operon the4′-phosphopantetheinyl-transferase is encoded. Although theantimicrobial activity could only be detected under iron-limitedconditions, no obvious fur-box could be identified within the operon,indicating a rather indirect effect of lack of iron on the expression ofthe operon.

This so-called lug operon was exclusively found in S. lugdunensis andencodes a unique combination of antibiotic biosynthesis enzymes, allwith less than 35% identity to any other described enzyme, suggestingthat it may be responsible for biosynthesis of a novel compound. Toconfirm that the lug operon is responsible for the antimicrobialactivity of IVK28, the smallest NRPS gene, lugD, was deleted by genereplacement. The mutant ΔlugD showed no detectable antimicrobialactivity, but the phenotype was restored by complementation with aplasmid-encoded copy of lugD (FIG. 1).

Computational analysis (NRPS predictor 2, Anti-Smash, HMMER) of theproteins encoded by lugA-D revealed an uncommon domain architecture,which is shown in FIG. 2. Most NRPS-systems assemble activated aminoacids in a linear fashion until product release, which is usuallycatalyzed by a type I thioesterase. In contrast, the S. lugdunensisoperon exhibits a putative reductase domain for final product release,encoded at the end of the lugC. At the same time, differing from LugA-C,LugD lacks a condensation domain, which is a common feature for socalled initiation modules that provide the first amino acids in someNRPS systems. The subsequent condensation reaction is then catalyzed byone of the elongation modules.

Adenylation-domain specificity prediction with the NRPS predictor 2 andAnti-Smash software gave valine and threonine (LugA), leucine (LugB),valine (LugC) and cysteine (LugD) as the most likely activated aminoacids, although with different probabilities (60% for Thr and 100% forCys).

3. Identification of the NRPS-II Product

Since the inhibitory activity against S. aureus only was detectable onagar plates containing 200 μM 2,2′-bipyridine, the same conditions wereused for an extraction attempt of the peptide. After growth for 48 hourscells of S. lugdunensis IVK28 were scraped off the agar and extractedwith 100% ethanol. Subsequent HPLC analysis of extracts from thewild-type and the M1 mutant strain revealed differences in only one mainpeak at 10.6 min retention time (with a molecular mass of 782.5 Da(FIGS. 3 A and B). Surprisingly, the absorbance spectrum showedcharacteristics of tryptophan containing proteins at 280 nm, although nosuch amino acid has been predicted according to the genetic organizationin silico. To confirm that the absence of the respective HPLC-peak is infact due to the integration of the transposon in lugA and not because ofan unidentified second-site mutation, a defined knock-out strain of lugDwas constructed (ΔlugD::erm). As expected, the HPLC chromatogram of therespective ΔlugD -mutant was nearly identical to the one of S.lugdunensis M1 and did not show the peak at 10.6 min (data not shown).The production phenotype could partially be restored by complementationwith the plasmid pRB474-lugD.

4. S. lugdunensis IVK28 is Able to Eliminate S. aureus USA300 inCo-Cultivation Experiments

To investigate if the expression of the NRPS-II system gives S.lugdunensis a competitive advantage, strains IVK28 or ΔlugD::erm wereco-cultivated in varying ratios with S. aureus USA300 on 2,2′-bipyridinecontaining agar plates. As shown in FIG. 4, S. lugdunensis IVK28 wasable to completely eradicate S. aureus USA300 from the mixture within 72hours, even with 90% S. aureus at the starting conditions (A). Incontrast, the mutant S. lugdunensis ΔlugD::erm only slightly decreasedthe S. aureus ratio within the first 48 hours and was subsequentlyovergrown by S. aureus within 72 hours (B). Even more, S. aureus wasable to displace the mutant when the starting conditions contained 90%S. lugdunensis (C). These results clearly demonstrate the importance ofthe NRPS-II system as a fitness-factor of S. lugdunensis in the struggleagainst S. aureus. Of note, the results were nearly identical when2,2′-bipyridine was omitted from the medium, indicating thatiron-limitation might not be the true and only inducing signal, butrather a special kind of stress like the close contact with S. aureus.

5. Overproduction and Purification of the NRPS-II Peptide

Interestingly, no antimicrobial activity could be detected, when S.lugdunensis IVK28 was grown in 2,2′-bipyridine containing liquidcultures, neither in cell extracts nor in the culture supernatant. Forthis reason the strain was genetically engineered by replacing thetetR-family like repressor gene upstream of lugA with thewell-established xylose-inducible xylR-regulatory system. This enabledthe inventors to induce peptide production by the addition of 0.5%xylose and in the absence of bipyridine. The corresponding strainΔtetR::erm/xylR exhibited significant antimicrobial activity in theculture supernatant after xylose addition. By 1-butanol extraction theinventors were able to concentrate the antimicrobial activity in thesolvent. After evaporation of 1-butanol, resuspension in 100% methanoland size exclusion chromatography on a Sephadex LH-20 column a highlyenriched active fraction could be obtained. Final purification wasperformed by preparative HPLC, resulting in the pure antimicrobialcompound, which was solved and stored in DMSO at a concentration of 10mg/ml at −20° C. LC-MS and MS-MS analysis confirmed the beforeascertained molecular weight of 782.5 Da with the elemental formulaC₄₀H₆₂N₈O₆S (FIG. 5).

An embodiment of the compound according to the invention named“lugdunin” was isolated as a white solid, the UV spectrum pointed to anindole ring and HR-ESI-MS revealed an ion peak at m/z=783.4581 ([M+H]⁺)and specific fragments in HPLC-MS-MS. Marfey's modification products ofD- and L-amino acid standards and of lugdunin were also subjected toHPLC-ESI-MS and HPLC-MS-MS. Mass adducts and fragments revealed D- andL-amino acids corresponding to three valine, leucine/isoleucine,tryptophan and a novel fragment for C₈H₁₅N₂OS assigned to a thiazolidinering structure. The ¹H-NMR spectrum showed characteristic aliphaticsignals for the valine protons and characteristic signals for thetryptophan moiety. The NMR spectra pointed to at least two differentisomers and conformers for lugdunin due to altered chemical shifts after48 hours in solution. Additional 2D NMR experiments supported thestructure of lugdunin. However, overlapping signals did not allow forthe full determination of the regiochemistry by NMR methods nor thestereochemistry of the single amino acids of lugdunin, the order of theD- and L-valine residues is not assignable via NMR spectroscopy.Therefore, lugdunin is assigned to the cyclic peptide depicted in FIGS.5A and B generated from the 7 amino acids and the empirical formulaC₄₀H₆₂N₈O₆S.

6. The New Compound is Bactericidal and Mainly Active Against MajorHuman Pathogens

To determine the spectrum of activity, a range of clinically relevantGram-positive and Gram-negative bacteria were used forMIC-determination. As shown in Table 1, it could be confirmed thatbeside various S. aureus strains the new compound is active against alltested species, including the glycopeptide-intermediate resistant S.aureus (GISA) and vancomycin resistant (VRE) Enterococcus faecalis andE. faecium, Streptococcus pneumoniae and Listeria monocytogenes. MICsranging from 1.5 to 12 μg/ml (1.9 to 15 μM) underscore the strongantibacterial potential of lugdunin. Interestingly, the S. aureus USA300MRSA strain was more susceptible than the laboratory strain RN4220. Theproducer strain showed also activity against Propionibacterium acnes,Streptococcus pyogenes, Micrococcus luteus and a range of otherstaphylococci. None of the Gram-negative bacteria was significantlyinhibited in the investigated concentration range (up to 100 μg/ml).

TABLE 1 MIC determination of lugdunin against various bacteria; MRSA,methicillin-resistant S. aureus; GISA, glycopeptide-resistant S. aureus;VRE, vancomycin-resistant enterococci STRAIN MIC ResistanceStaphylococcus aureus USA 300 (LAC) 1.5 μg/ml MRSA Staphylococcus aureusUSA300 (NRS384) 1.5 μg/ml MRSA Staphylococcus aureus Mu50 3 μg/ml GISAStaphylococcus aureus SA113 3 μg/ml Staphylococcus aureus RN4220 3 μg/mlEnterococcus faecalis VRE366 12 μg/ml VRE Enterococcus faecium BK463 3μg/ml VRE Listeria monocytogenes ATCC 19118 6 μg/ml Streptococcuspneumoniae ATCC 49619 1.5 μg/ml Pseudomonas aeruginosa PAO1 >50Escherichia coli DH5α >50

Lugdunin was bactericidal against MRSA with complete killing after asingle dose treatment (FIG. 6A). No spontaneous resistance developmentwas observed in S. aureus during continuous subcultivation of S. aureuswith subinhibitory concentrations of lugdunin over 30 days (FIG. 6B). Incontrast, treatment with rifampicin led to rapidly increasingspontaneous resistance within a few days (FIG. 6B).

To test whether the activity is bacteriostatic or bactericidal, killingassays were performed with S. aureus USA300 and peptide concentrationsof 1×MIC (1.5 μg/ml). As shown in FIG. 7, a reduction of viable cells ofat least two log units was achieved within 6 hours incubation in PBS,clearly indicating a bactericidal mode of action.

7. Topical Treatment with Lugdunin is Effective in an In Vivo MouseModel

Since the effectiveness of lugdunin could be shown in vitro, the nextstep was the development of an in vivo model. For this purpose the socalled tape stripping model was applied [Wanke et al. (2013)Staphylococcus aureus skin colonization is promoted by barrierdisruption and leads to local inflammation. Exp Dermatol 22: 153-15].For this model the back of C57BL/6 mice was shaved and the skin barrierwas disrupted by strong tape-stripping (7 times) without creatingwounds. S. aureus Newman (inoculum of 10⁷ cfu in 15 μl phosphatebuffered saline (PBS)) was applied on the disrupted skin and covered byFinn Chambers for 20 hours to ensure efficient colonization. Due to itshydrophobic nature lugdunin was solved in 100% DMSO to a concentrationof 10 mg/ml and subsequently diluted into 100% sesame oil to a finalconcentration of 100 μg/ml. 15 μl of this lugdunin preparation wereapplied to the colonized spots 18, 24, and 42 hours after theapplication of S. aureus. For the control only 1% DMSO in sesame oil wasapplied. Three hours after the final application mice were sacrificedand skin biopsy punches were analyzed for the presence of S. aureus. Theinventors distinguished between the washing fraction (loosely attachedbacteria removed by a washing step in PBS) and the scrape fraction(destruction of the skin material with scalpels to release bacteria fromdeep skin areas). FIG. 8 clearly shows the reduction of S. aureus bylugdunin treatment after 45 hours in the wash fraction as well as in thescrape fraction, indicating that lugdunin is also penetrating deepertissue areas.

In a preliminary experiment, where the lactate-dehydrogenase release ofneutrophil granulocytes was measured, no significant cytotoxicity couldbe observed within 3 h incubation of the cells with the peptide, even atconcentrations of 50 μg/ml resembling a more than 30-fold MIC for S.aureus USA300; see FIG. 9.

8. Lugdunin Production Outcompetes S. aureus

The production of antimicrobials, mostly plasmid-encoded ribosomallysynthesized bacteriocins, has been sporadically documented in individualbacterial strains from human microbiomes. However, the roles of suchcompounds in microbial fitness and in microbiota dynamics have remainedlargely unknown. To determine whether lugdunin contributes to thecapacity of S. lugdunensis IVK28 to prevail in competition with S.aureus, the two species were co-cultivated on solid agar surface,promoting lugdunin production, and bacterial numbers were monitored forthree days.

As shown in FIG. 10A, the lugdunin-producing IVK28 wild type overgrew S.aureus efficiently, even when the inoculum contained 10-times highernumbers of S. aureus than S. lugdunensis cells. No viable S. aureuscells were recovered after three days indicating complete killing by S.lugdunensis. In contrast, IVK28 ΔlugD was overgrown by S. aureus evenwhen it was inoculated at 10-times higher numbers than S. aureus (FIGS.10 B, D). The S. aureus-eradicating capacity of ΔlugD could be largelyrestored by complementation with lugD on a plasmid (FIG. 10 C). Thesedata demonstrated that S. lugdunensis can effectively eradicate S.aureus and that lugdunin production is responsible for this trait.

Nasal carriage is known to be a major risk factor for invasive S. aureusinfections. To explore whether S. lugdunensis can interfere with nasalS. aureus colonisation in vivo in vertebrates, the noses of cotton rats,a well-established animal model for investigating S. aureus nasalcolonisation, were instilled with mixtures of S. lugdunensis IVK28 wildtype or ΔlugD plus S. aureus. The three test strains colonised cottonrat noses stably over the 5-day test period when instilled individually(FIG. 11). However, when the two species were co-inoculated,significantly less S. aureus cells were retrieved from animalsco-colonised by IVK28 wild type compared to those co-colonised withΔlugD (FIG. 10 E). This finding indicates that lugdunin production caneffectively interfere with S. aureus colonisation in vivo.

9. The Presence of S. lugdunensis in the Human Nose is Very LikelyCorrelated with the Absence of S. aureus

As a proof of principle of the influence of S. lugdunensis on S. aureuswe investigated the co-occurrence of the two species in human noses. Forthis, nasal swabs of 187 risk patients were investigated. In total 61individuals were colonized with S. aureus (32.6%) and 17 with S.lugdunensis (9.1%). None of the species was found in 109 people (58.2%).From these numbers 2.97% (nearly 6 individuals) can be expected to beco-colonized with S. aureus and S. lugdunensis. In contrast, only oneperson was identified being co-colonized, which is significantly lessthan expected (FIG. 12, p<0.05). This might indicate that in vivo thepresence of S. lugdunensis inhibits the colonization with S. aureus bythe secretion of lugdunin. In line with this assumption is the detectionof the mass of lugdunin (782.4513 Da) in butanol extracted swabs of S.lugdunensis carriers, but not of non-carriers (data not shown).

10. Biological Activity of Lugdunin Derivatives

The inventors have synthesized numerous chemical derivatives of lugduninto evaluate an abstract or general chemical formula representing theprototype of the newly found anti-bacterial activity. The chemicalderivatives are shown in the subsequent tables. All syntheticderivatives were tested for biological activity against S. aureusUSA300. Measured values resulting therefrom were classified and markedwith “+”. Derivatives marked with “+” result in the respectiveconcentration range a complete inhibition of the bacterial growth, i.e.no growth. The categories are evident from the following Table 2.

TABLE 2 MIC in pg/ml 200 100 50 25 12.5 1.5 − + ++ +++ ++++    inactiveslightly active active very active

Thus, the indication of a biological activity with “++++” means a MICbetween 1.5 and 12.5 μg/ml. Lugdunin is the references substance withthe highest biological activity and a MIC of 1.5 μg/ml. In addition toderivatives resulting in a complete killing of the USA300 test strainderivatives were synthesized which induce a significant growthreduction. They do not result in a complete killing of all bacterialcells of the test strain. Such derivatives are marked with “♦”.

TABLE 3 Formula (I)

Variable cycle sizes and hetero atoms in the thiazolidin cycle. m = 1and 2; n = 0, 1; Y = S, O, CH₃ (special case upper right box) 1-L-Cys2-D-Val 3-L-Trp 4-D-Leu 5-L-Val 6-D-Val 7-D-Val ⇐ Sequence of thenatural substance Bioactivity against S aureus USA 300 X #1 ⬇ For ″m″ ⬇1-L- 2-D-Val 3-L-Trp 4-D-Leu 5-L-Val 6-D-Val 7-L-Val ♦ Homoserine For″n″ ⬇ 1-L-Cys ▪ 3-L-Trp 4-D-Leu 5-L-Val 6-D-Val 7-L-Val ♦ 1-L-Cys ▪3-L-Trp 4-D-Leu 5-L-Leu 6-D-Leu 7-L-Val + For ″Y″ ⬇ 1-L-Cys 2-D-Val3-L-Trp 4-D-Leu 5-L-Val 6-D-Val 7-L-Val ++++ 1-L-Ser 2-D-Val 3-L-Trp4-D-Leu 5-L-Val 6-D-Val 7-L-Val ++ Special case ⬇ 1-L-Ala 2-D-Val3-L-Trp 4-D-Leu 5-L-Val 6-D-Val 7-L-Val ♦

TABLE 4 Formula (II)

1-L-Cys 2-D-Val 3-L-Trp 4-D-Leu 5-L-Val 6-D-Val 7-D-Val ⇐ Sequence ofthe natural substance X #1 X #2 X #3 X #4 X #5 X #6 X #7 Bioactivityagainst S ⬇ ⬇ ⬇ ⬇ ⬇ ⬇ ⬇ aureus USA 300 X #1 ⬇ 1-L-Ala 2-D-Val 3-L-Trp4-D-Leu 5-L-Val 6-D-Val 7-L-Val + 1-L-Cys 2-D-Ala 3-L-Trp 4-D-Leu5-L-Val 6-D-Val 7-L-Val +++ 1-L-Cys 2-D-Leu 3-L-Trp 4-D-Leu 5-L-Val6-D-Val 7-L-Val ++++ 1-L-Cys 2-L-Val 3-L-Trp 4-D-Leu 5-L-Val 6-D-Val7-L-Val ++ 1-L-Cys 2-D-Val 3-L-Ala 4-D-Leu 5-L-Val 6-D-Val 7-L-Val ♦1-L-Cys 2-D-Val 3-L-DOPA 4-D-Leu 5-L-Val 6-D-Val 7-L-Val ♦ 1-L-Cys2-D-Val 3-L-Tyr 4-D-Leu 5-L-Val 6-D-Val 7-L-Val + 1-L-Cys 2-D-Val3-L-Phe 4-D-Leu 5-L-Val 6-D-Val 7-L-Val +++ 1-L-Cys 2-D-Val 3-L-Trp4-D-Ala 5-L-Val 6-D-Val 7-L-Val ♦ 1-L-Cys 2-D-Val 3-L-Trp 4-D-Leu5-L-Ala 6-D-Val 7-L-Val +++ 1-L-Cys 2-D-Val 3-L-Trp 4-D-Leu 5-L-Thr6-D-Val 7-L-Val ++ 1-L-Cys 2-D-Val 3-L-Trp 4-D-Leu 5-L-Val 6-D-Ala7-L-Val ++ 1-L-Cys 2-D-Val 3-L-Trp 4-D-Leu 5-L-Val 6-D-Thr 7-L-Val +1-L-Cys 2-D-Val 3-L-Trp 4-D-Leu 5-L-Val 6-D-Trp 7-L-Val ++++ 1-L-Cys2-D-Val 3-D- 4-D-Leu 5-L-Val 6-D-Trp 7-L-Val ♦ Anthranyl- Ala 1-L-Cys2-D-Val 3-L-Ala 4-D-Leu 5-L-Val 6-D-Trp 7-L-Val + 1-L-Cys 2-D-Val3-L-Val 4-D-Val 5-L-Leu 6-D-Trp 7-L-Val ++++ 1-L-Cys 2-L-Val 3-D-Val4-L-Val 5-D-Leu 6-L-Trp 7-L-Val +++ 1-D-Cys 2-L-Val 3-D-Trp 4-L-Leu5-D-Val 6-L-Val 7-L-Val +++

TABLE 5 Formula (III)

Modification: Free stereochemistry and variable position 2 1-L-Cys2-D-Val 3-L-Trp 4-D-Leu 5-L-Val 6-D-Val 7-D-Val ⇐ Sequence of thenatural substance Y #2 Bioactivity against S ⬇ aureus USA 300 X #1 ⬇1-L-Cys 2-D-Leu 3-L-Trp 4-D-Leu 5-L-Val 6-D-Val 7-L-Val ++++ 1-L-Cys2-D-Ala 3-L-Trp 4-D-Leu 5-L-Val 6-D-Val 7-L-Val +++ 1-L-Cys 2-L-Val3-L-Trp 4-D-Leu 5-L-Val 6-D-Val 7-L-Val ++ 1-D-Cys 2-L-Val 3-D-Trp4-L-Leu 5-D-Val 6-L-Val 7-L-Val +++ 1-L-Cys 2-L-Val 3-D-Val 4-L-Val5-D-Leu 6-L-Trp 7-L-Val +++

TABLE 6 Formula (IV)

Lugdunin naural substance as it is isolated from S. Lugdunensis (MIC =1.5 μg/ml) 1-L-Cys 2-D-Val 3-L-Trp 4-D-Leu 5-L-Val 6-D-Val 7-L-Val ++++

From theses experiments the inventors were able to identify an abstractor general chemical formula representing the prototype of the newlyfound anti-bacterial activity. Such formula is depicted in claim 1. Thesubstituents m, n, X, and Y can be varied within the indicated rangeswithout losing the anti-bacterial activity, as it is demonstrated bythese experiments.

9. Summary

Here the inventors describe the isolation and structure elucidation ofthe novel bactericidal peptide antibiotic lugdunin, which is activeagainst S. aureus and other pathogenic bacterial species. On the basisof this peptide derivatives have been synthesized and tested for theiranti-bacterial activity. As a result, a core structure has beendeveloped by the inventors, which exhibits the observed activity.

The isolated naturally occuring peptide is non-ribosomally produced by aStaphylococcus lugdunensis isolate (strain IVK28) where thecorresponding NRPS-operon is chromosomally encoded. However, genomedatabase analysis and PCR-amplification experiments with 14 naturalisolates indicated that the operon is present in all investigatedstrains, although not all of them exhibited the antibiotic activity(data not shown). Except for the description of micrococcin P1production in the single animal associated Staphylococcus equorum strainWS2733 (Carnio et al. (2001) Pyridinyl polythiazole class peptideantibiotic micrococcin P1, secreted by foodborne Staphylococcus equorumWS2733, is biosynthesized nonribosomally. Eur J Biochem 268: 6390-6401),no NRPS-peptides are known for the genus Staphylococcus that exhibitantibacterial properties. Micrococcin P1 was originally identified inMicrococcus varians and Bacillus pumilus, but lugdunin represents thefirst genus-specific antibacterial NRPS product with a novel structurefor Staphylococci. Since S. lugdunensis can be frequently isolated fromthe human nose, it is a potential competitor of S. aureus in thishabitat. The inventor's co-cultivation experiments have clearly shownthat the production of lugdunin equips S. lugdunensis with a strongadvantage in competition. Even a minority of S. lugdunensis IVK28 cellsat the starting conditions can eradicate S. aureus from the culturewithin 72 hours. Also, purified lugdunin is effective in eradication ofS. aureus in a mouse model (tape stripping model).

Lugdunin represents a novel and rather uncommon structure since itcomprises a tryptophan residue in combination with three consecutivevaline residues, of which one is part of a valinoyl-thiazolidine ringstructure. Tryptophan and the thiazolidine portion are flanking a fourthvaline residue. A high content of alternating D- and L-valine has beenfound in the macrolactone antibiotic valinomycin, which acts as anionophor, but there is no structural similarity to lugdunin. A combinedL-tryptophan-thiazole structure has been described for the proteinsynthesis inhibitors A21459 [Ferrari et al. (1996) Antibiotics A21459 Aand B, new inhibitors of bacterial protein synthesis. II. Structureelucidation. J Antibiot (Tokyo) 49: 150-154], Kocurin [Martin et al.(2013) Kocurin, the true structure of PM181104, ananti-methicillin-resistant Staphylococcus aureus (MRSA) thiazolylpeptide from the marine-derived bacterium Kocuria palustris. Mar Drugs11: 387-398], or the 7-methoxy-tryptophan containing zelkovamycin[Tabata N, Tomoda H, Zhang H, Uchida R, Omura S (1999) Zelkovamycin, anew cyclic peptide antibiotic from Streptomyces sp. K96-0670. II.Structure elucidation. J Antibiot (Tokyo) 52: 34-39]. Nevertheless,there is no additional similarity between lugdunin and the mentionedantibiotics, which makes the target prediction for lugdunin purelyspeculative. Since lugdunin exhibits bactericidal activity, its mode ofaction might differ from the other described peptides, whose activity isbacteriostatic.

Beside the use of purified peptide and derivatives thereof foreradication strategies also the preventive application of a lugduninproducer strain might be possible to e.g. clear S. aureus colonizationin human noses.

Therefore, what is claimed, is:
 1. Compound of the formula of:

wherein X is selected from the group consisting of: H, CH₃, CH₂CH₃,

under the proviso that at least one X is:

and Y is selected from the group consisting of O, S, and NH, m is aninteger from 1 to 3, n is an integer from 0 to 4, and the salts thereof,the solvates thereof and the solvates of the salts thereof, furtherunder the proviso that the compound is not a compound of formula (III)

wherein Y1 is selected from the group consisting of

and further under the proviso that when m is 1, n is 2, and at least oneX is

then at least two X are


2. Compound of claim 1 comprising the following formula:

wherein X is selected from the group consisting of: H, CH₃, CH₂CH₃,anthranylalanine, DOPA, tyrosine, threonine,

under the proviso that at least one X is:


3. Pharmaceutical composition comprising the compound of claim 1 and apharmaceutically acceptable carrier.
 4. Method for the treatment orprophylaxis of a disease in a living being, comprising theadministration to said living being of an antibacterially effectiveamount of the compound of formula (I),

wherein X is selected from the group consisting of: H, CH₃, CH₂CH₃,

under the proviso that at least one X is:

and Y is selected from the group consisting of O, S, and NH, m is aninteger from 1 to 3, n is an integer from 0 to 4, and the salts thereof,the solvates thereof and the solvates of the salts thereof, wherein thedisease is selected from one or more members in the group of: infectiousdisease, bacterial disease, infection by a Gram-positive bacterium,infection by Staphylococcus aureus, infection by methicillin-resistantStaphylococcus aureus (MRSA), and infection by vancomycin-resistantStaphylococcus aureus (VRSA).
 5. Method of producing the compound offormula (IV)

comprising the following steps: (1) Providing bacteria of the species ofStaphylococcus lugdunensis, (2) Purifying the compound of formula (IV)from said bacteria.
 6. The method of claim 5, wherein after step (1) andbefore step (2) the bacteria and a medium for the cultivation of thebacteria are extracted and the bacteria and medium extract are subjectedto step (2) where the compound is purified from said bacteria and mediumextract.
 7. Method of claim 6, wherein in step (2) the purificationinvolves the use of high performance liquid chromatography (HPLC)identifying a signal peak associated with said compound.
 8. Method ofclaim 7, wherein said signal peak corresponds to a molecular mass whichis selected from the group of ranges of molecular masses consisting of:approx. 650 Da-950 Da, approx. 700 Da-850 Da, approx. 750 Da-800 Da,approx. 770 Da-790 Da, and approx. 782.5 Da.
 9. Method of producing thecompound of formula (IV)

comprising the following steps: (1) Expressing the non-ribosomal peptidesynthetase system II of the species of Staphylococcus lugdunensis(NRPS-II) in a biological system, (2) Incubating the expressed NRPS-IIunder conditions allowing the synthesis of the compound of formula (IV),(3) Purifying said compound.
 10. Method of claim 9, wherein said NRPS-IIis encoded by a nucleic acid molecule comprising any of the codingsequences of the genes lugA, lugB, lugC, and lugD.
 11. Method of claim10, wherein said nucleic acid molecule further comprises at least oneelement selected from the group consisting of: transcriptional regulatorof GntR family, ABC transporter, ABC-2 type transport system permeaseprotein, hyp. membrane protein, TetR/AcrR family regulator, Thioesterasefamily protein, 4′-phosphopantetheinyltransferase, and put. negativeregulator of sigY.
 12. Method of claim 10, wherein the conditionscomprise amino acids, thioesterase, and buffer.
 13. A pharmaceuticallyacceptable composition comprising a compound of the formula (I):

wherein X is selected from the group consisting of: H, CH₃, CH₂CH₃,

under the proviso that at least one X is:

and Y is selected from the group consisting of O, S, and NH, m is aninteger from 1 to 3, n is an integer from 0 to 4, and further under theproviso that the compound is not the compound of formula (IV)

and the salts thereof, the solvates thereof and the solvates of thesalts thereof, and a pharmaceutically acceptable carrier.
 14. Thecomposition of claim 13, wherein the compound is according to formula(II):

wherein X is selected from the group consisting of: H, CH3, CH2CH3,anthranylalanine, DOPA, tyrosine, threonine,

under the proviso that at least one X is:


15. The composition of claim 13, wherein the compound is according toformula (III):

wherein Y1 is selected from the group consisting of:


16. The method of claim 4, wherein the compound is according to formula(III):

wherein Y1 is selected from the group consisting of:


17. The method of claim 4, wherein the compound is the compound offormula (IV):


18. The method of claim 4, wherein the disease is infection by aGram-positive bacterium.
 19. The method of claim 4, wherein the diseaseis infection by Staphylococcus aureus.
 20. The method of claim 4,wherein the disease is infection by methicillin-resistant Staphylococcusaureus (MRSA).
 21. The method of claim 4, wherein the disease isinfection by vancomycin-resistant Staphylococcus aureus (VRSA).
 22. Themethod of claim 17, wherein the disease is infection by a Gram-positivebacterium.
 23. The method of claim 17, wherein the disease is infectionby Staphylococcus aureus.
 24. The method of claim 17, wherein thedisease is infection by methicillin-resistant Staphylococcus aureus(MRSA).
 25. The method of claim 17, wherein the disease is infection byvancomycin-resistant Staphylococcus aureus (VRSA).
 26. A compound of theformula of:

and the salts thereof, the solvates thereof and the solvates of thesalts thereof.
 27. Pharmaceutical composition comprising the compound ofclaim 26 and a pharmaceutically acceptable carrier
 28. A compound of theformula of

and the salts thereof, the solvates thereof and the solvates of thesalts thereof.
 29. Pharmaceutical composition comprising the compound ofclaim 27 and a pharmaceutically acceptable carrier.
 30. Method for thetreatment or prophylaxis of a disease in a living being, comprising theadministration to said living being of an antibacterially effectiveamount of the compound of formula (V),

wherein the disease is selected from one or more members in the groupof: infectious disease, bacterial disease, infection by a Gram-positivebacterium, infection by Staphylococcus aureus, infection bymethicillin-resistant Staphylococcus aureus (MRSA), and infection byvancomycin-resistant Staphylococcus aureus (VRSA).
 31. Method for thetreatment or prophylaxis of a disease in a living being, comprising theadministration to said living being of an antibacterially effectiveamount of the compound of formula (VI),

wherein the disease is selected from one or more members in the groupof: infectious disease, bacterial disease, infection by a Gram-positivebacterium, infection by Staphylococcus aureus, infection bymethicillin-resistant Staphylococcus aureus (MRSA), and infection byvancomycin-resistant Staphylococcus aureus (VRSA).